High fat diet is associated with gut microbiota dysbiosis and decreased gut microbial derived metabolites related to metabolic health in young Göttingen Minipigs

The objectives were 1) to characterize a Göttingen Minipig model of metabolic syndrome regarding its colon microbiota and circulating microbial products, and 2) to assess whether ovariectomized female and castrated male minipigs show similar phenotypes. Twenty-four nine-week-old Göttingen Minipigs were allocated to four groups based on sex and diet: ovariectomized females and castrated males fed either chow or high-fat diet (HFD) for 12 weeks. At study end, body composition and plasma biomarkers were measured, and a mixed meal tolerance test (MMT) and an intravenous glucose tolerance test (IVGTT) were performed. The HFD groups had significantly higher weight gain, fat percentage, fasting plasma insulin and glucagon compared to the chow groups. Homeostatic model assessment of insulin resistance index (HOMA-IR) was increased and glucose effectiveness derived from the IVGTT and Matsuda´s insulin sensitivity index from the MMT were decreased in the HFD groups. The HFD groups displayed dyslipidemia, with significantly increased total-, LDL- and HDL-cholesterol, and decreased HDL/non-HDL cholesterol ratio. The colon microbiota of HFD minipigs clearly differed from the lean controls (GuniFrac distance matrix). The main bacteria families driving this separation were Clostridiaceae, Fibrobacteraceae, Flavobacteriaceae and Porphyromonadaceae. Moreover, the species richness was significantly decreased by HFD. In addition, HFD decreased the circulating level of short chain fatty acids and beneficial microbial metabolites hippuric acid, xanthine and trigonelline, while increasing the level of branched chain amino acids. Six and nine metabolically relevant genes were differentially expressed between chow-fed and HFD-fed animals in liver and omental adipose tissue, respectively. The HFD-fed pigs presented with metabolic syndrome, gut microbial dysbiosis and a marked decrease in healthy gut microbial products and thus displayed marked parallels to human obesity and insulin resistance. HFD-fed Göttingen Minipig therefore represents a relevant animal model for studying host-microbiota interactions. No significant differences between the castrated and ovariectomized minipigs were observed.


Animal study procedures for castration and ovariectomized
Twelve young male and 12 young female Göttingen Minipigs (Ellegaard Göttingen Minipigs A/S, Dalmose, Denmark).The health status of the pigs can be found on Ellegaard Göttingen Minipigs home page: https://minipigs.dk/about-gottingen-minipigs/health-status.The pigs were either castrated or ovariectomized (OVX) 11 days prior to study start at which point they were 9 weeks of age.Before surgery the pigs were acclimated for 9 days to the facility.
Anaesthesia was induced with Zoletil-mixture for minipigs in a dose of 0.1 ml/kg given i.m.
After the pig was anaesthetised an injection of 1 mg atropine/mL (0.02 mL/kg) was given i.m.For ovariectomy, the pig was hereafter intubated, and the anaesthesia was maintained on a mixture of isoflurane and oxygen.For castration, the procedure was quick and performed under the Zoletil mix anaesthesia.
The surgical region was shaved, washed thoroughly with soap and water and disinfected with 70 % ethanol or iodine, where after sterile drapes were used to secure aseptic access to the entire surgery field.
For ovariectomy, the ovaries were localised and isolated by placing a haemostatic forceps on the salpinx and ovarian artery without damaging the uterine horn.Then the ovaries were cut off using ultrasonic shears (Harmonic ACE®).After inspection to ensure proper haemostasis and removal of all ovarian tissue, the procedure was repeated on the second ovary.Lastly, the abdominal incision was closed in 2-3 layers.For castration, a scrotal incision was made over the testicle through the skin, subcutaneous tissue and tunica vaginalis to expose the testicle.The spermatic cord was cut by ultrasonic shears (Harmonic ACE®) assuring proper haemostasis.After repeating the procedure on the second testicle, the incision was closed in 2-3 layers.

Implantation of the central-venous catheter
Central venous catheters were implanted through an ear vein after approx.11 weeks of diet feeding (Day 78-80).During the catheter implantation, the pigs were anaesthetized i.m. with 1 mL/10 kg of the Zoletil mixture described above.In addition, the pigs were given an i.m. injection of 1 mg atropine/mL (0.01 mL/kg).A central venous catheter (BD Careflow 3Fr 200mm, Argon Medical, Texas, USA) was implanted in v. jugularis via an ear vein using a minimally invasive technique.The catheter was fixed using a special plate and steel piercings.After implantation of the catheter and the piercings and on the following two days, the pigs were treated i.m. with 0.4 mg/kg meloxicam (Metacam®, 20 mg/ml).The catheter was used for stress-free blood sampling during the metabolic tests.

Mixed meal test (MMT)
The mixed meal was prepared as follows: The same amount of each of the following ingredients were mixed: Skyr (Løgismose, 10 g protein/100 g), double cream (38 g fat/100 g) and powdered sugar (100 g sugar/100 g).The pigs were fed 6 g/kg of the mixture, resulting in the following doses of macronutrients: 0.24 g protein/kg, 0.76 g fat/kg and 2.1 g glucose/kg and the clock was started when the meal was finished.Blood (2 mL) was sampled at the following time points in relation to the meal: Pre-dose and 15, 30, 45, 60, 90, 120, 150 and 180 minutes.The blood samples were transferred to EDTA tubes with 25 μl special stabilization buffer/ml blood (50 սl in total for the 2 ml blood) (see below for description if the stabilization buffer).

Gene expression analysis
RNA was extracted from ~30 mg of liver using the RNeasy Mini Kit (Qiagen), from 100 mg of omental fat tissue using the protocol of (1) and from 100 mg skeletal muscle tissue using the RNeasy Fibrous Tissue Mini Kit (Qiagen).The GentleMACS TM Octo Dissociator machine (Milteny Biotec) with M tubes was used for homogenization.All samples were DNAse treated according to each protocol.100 ng of each RNA sample were used to make 2 cDNA replicates using ImProm II reverse transcriptase (Promega, Denmark) and a 3:1 mixture of random hexamers/OligodT primers, according to the manufacturer's instructions.cDNA from liver was diluted 16 times, while cDNA from omentum and muscle were diluted 8 times before using in quantitative real-time PCR (qPCR).
Quantity and quality of each RNA sample were assessed by NanoDrop TM spectrophotometer machine (Thermo Scientific) and by the RNA 6000 Nano Kit (Agilent Technologies) in an Agilent RNA Bioanalyzer system (Agilent Technologies) respectively.Only RQI values of >6 were considered acceptable (the average values for each tissue were as follows: liver -8.43 ± 0.23; omental fat tissue -7.72 ± 0.66 (one pig from the female chow group was excluded due to a low RIN value); skeletal muscle tissue -9.07 ± 0.11).Subsequently, cDNA was synthesized from 100ng of total RNA using ImPromII reverse transcriptase (Promega), RNAsin (Promega) and oligodT/random hexamers (1:3) primers following manufacturer's recommendations.Two cDNA replicates were made from each RNA sample to account for technical variation.A negative control (-RT; without reverse transcriptase) was made for each tissue to test the presence of genomic DNA.cDNA from liver was diluted 16 times, while cDNA from omentum and skeletal muscle was diluted 8 times before using in quantitative real-time PCR (qPCR).
A panel of 96 different obesity relevant genes including possible reference genes were chosen from an in-house primer library (Suppl.Table S2) for profiling by qPCR using the high-throughput qPCR Biomark HD platform (Fluidigm, San Francisco, California) following manufacturer's protocols.
The qPCR was performed using Flex Six IFC chips for assay optimization and 96.96 IFC chips in a Biomark TM HD machine (Fluidigm) using SsoFast TM EvaGreen ® following manufacturer instructions for reactions and thermal profile.